Microscopy Techniques

NCI Optical Microscopy Laboratory

Techniques

All of the following techniques are available in at least one of the core sites.
Please consult the individual core sites under Locations to see which techniques that core site offers.

Multicolor imaging

Fluorescence imaging is used for protein localization and colocalization in 3D. Multi-color imaging is necessary to observe colocalization of several proteins in the same cell. Many fluorescent proteins are now available for multi-color labeling and imaging of three to four different fluorophores is possible on most microscopes. Specialized microscopes are also available for imaging even more colors simultaneously. This can be accomplished by carefully designed filter combinations or by the technique of spectral unmixing. Spectral unmixing is based on an analysis of the optical spectra of different fluorophores. It allows discriminating dyes or fluorescent proteins with similar spectra, such as GFP and YFP. However, this technique has its limitations. If different labels in the specimen are vastly different in intensity, the spectral unmixing will not work properly.

Time Lapse Imaging

Live imaging of cells and tissues tagged with fluorescent markers provides a much deeper understanding of cellular processes than imaging of a fixed specimen, since live cell imaging permits analysis of the dynamics of biological processes. Time-lapse experiments typically investigate changes in fluorescently tagged protein localization over time, such as movement of a protein from the cytoplasm to the nucleus. These experiments are usually done on a microscope equipped with environmental chambers that regulate temperature and CO2 on the microscope stage. In addition, time-lapse studies are typically done with very sensitive cameras or detectors, since these allow low level excitation of the specimen which keeps it healthy.

FRAP and FCS

FRAP (Fluorescence Recovery After Photobleaching) and FCS (Fluorescence Correlation Spectroscopy) allow one to monitor and quantify the movement of tagged proteins. In FRAP the molecules contained in a specific region are “turned off” by means of photobleaching. If molecules are free to move, the exchange between bleached and non-bleached molecules results in the recovery of fluorescence within the photobleached region. The recovery data can be analyzed to provide information about the diffusion and binding rates of the tagged protein. In FCS, the number of molecules present within a diffraction-limited spot are counted as a function of time. The rate of fluctuations in this number contains information about the mobility of the fluorescently tagged molecules, which as in FRAP can be used to deduce quantitative information about the rates of diffusion or binding.

FRET

FRET (Fluorescence Resonance Energy Transfer) is a technique to detect protein interaction and the formation of complexes between different proteins tagged with fluorescence markers. It also can be used to detect enzymatic activity by special reporters (FRET biosensors). There are several different approaches to detect FRET including sensitized emission or acceptor photobleaching. Sensitized emission measurements can be performed on either a conventional wide-field fluorescence microscope or on a confocal, whereas acceptor photobleaching measurements require a microscope equipped with a laser for photobleaching, present on all confocal microscopes.

Super-resolution microscopy

The conventional diffraction limit for visible light sets a resolution limit of ~200 nm when using a wide-field, confocal, deconvolution or two photon microscope. In the past decade, several pioneering approaches have been developed that bypass this limit. All are based in one way or another on the unique properties of fluorescence molecules that allow them to be switched on and off. There are now three different super-resolution approaches, STED, PALM and structured illumination. STED and PALM can achieve resolutions down to 10-20 nm in a two-dimensional image, although in general neither technique provides significantly improved z resolution. Structured illumination improves resolution in xy to ~100 nm, but also significantly improves z resolution.

Single Molecule Imaging

The PALM super-resolution approach can be easily adapted for detection of single molecules inside of cells. The motion of these individual molecules can be studied by collecting time lapse images. This can be used to measure the movement of molecules through the cytoplasm or nucleus. It can also be used to measure the binding residence times of molecules on relatively stable scaffolds such as chromatin or the cytoskeleton.

New Fluorescent Probes

There is an ever increasing supply of new fluorescent probes that are not only brighter than older probes but have various special features that can be useful in advanced applications. Many of these probes have been developed for live-cell imaging by fusion to a protein of interest. Some of the newer probes are not only brighter and more stable than GFP, but also provide many other colors to permit multi-color imaging. Other probes have special properties that allow them to be turned on and off or to be switched from one color to another color. Finally, there are also alternative protein fusion strategies now available that allow labeling of proteins with very stable organic fluorophores. These probes can be useful for long term time-lapse studies or single molecule detection.